Since their first identification in clinical specimen in a remote village in Papua New Guinea in 1967 penicillin resistant pneumococci have spread globally. Exceedingly high incidence of pneumococcal resistance has been reported (50% of all invasive isolates in Spain and 70% of all pediatric isolates in Hungary). Recent and alarming developments include: the appearance of resistance to third-generation cephalosporins; increase in the penicillin MIC and demonstration of the capacity of certain multiresistant clones to spread over large geographic distances, to cause a high proportion of upper respiratory pneumococcal infections, and to extensively colonize healthy children. In order to gain some control over the explosive spread of these dangerous community-acquired pathogens, it will be mandatory to better understand the molecular basis of beta-lactam antibiotic resistance; the mechanism(s) that control the level (MIC) of penicillin resistance and the origin and nature of genetic elements that define the complex pleiomorphic phenotypes of these bacteria. The list of these properties is quite long and the underlying mechanisms are unknown. At least 4 out of the 5 penicillin-binding proteins (PBPs) of highly resistant strains have reduced affinity for penicillin. Resistant isolates exhibit a high degree of polymorphism in their PBPs and contain PBP genes of "mosaic" structure. The great majority of penicillin resistant strains are restricted to only a few (4 to 6) of the 83 pneumococcal capsular types. Many resistant isolates produce cell wall peptidoglycans of grossly altered chemical structure. Inhibited or defective autolysis during penicillin treatment is frequently associated with resistance and some resistant isolates also show reduced sensitivity to the bactericidal effect of penicillin. We propose to refer to the genetic determinants of these phenotypes collectively as penicillin response genes. The purpose of this grant proposal is to identify and dissect these genes and to provide molecular level understanding for their functioning. Specifically: (A) We shall try to identify the minimal sequence alterations in the mosaic PBP genes that are associated with the reduced antibiotic affinity of the particular PBP and increased antibiotic resistance (MIC value) of the cells. (B) The same approach will be used to identify the genetic element(s) responsible for the altered chemical composition of peptidoglycan in resistant cells. These studies will be done in collaboration with Dr. Chris Dowson of the University of Sussex. (C) The great majority of penicillin resistant pneumococci also carry genetic traits related to the mechanism of irreversibility of penicillin action, since resistant isolates often show reduced rates of antibiotic-induced lysis and viability loss. Bacteria with similar phenotypes have recently been isolated in our laboratory by insertion/duplication mutagenesis and a key genetic determinant controlling bactericidal sensitivity of pneumococci has also been identified. A major effort will be made to clone these genes and to clarify biochemical (enzymatic) mechanisms involved. We anticipate that these approaches will provide information important for the control of the spread of antibiotic resistant strains. The studies should also allow novel insights into as yet unexplored areas of the molecular biology and genetics of cell wall synthesis in pneumococci and other bacteria.